Endothelial lipase (EL) is an extracellular protein with significant hydrolysis activity against HDL. Studies in model organisms suggest that EL is important in HDL metabolism;however, the significance of EL in human HDL metabolism is unknown. The objective of this proposal is to investigate variation in human EL and the effects of this variation on HDL levels. Deep resequencing of EL in subjects with high HDL has identified potentially functional nonsynonymous and promoter variants. We hypothesize that rare EL coding variants increase HDL by decreasing EL activity. We also hypothesize that EL promoter variants modulate HDL levels by affecting levels of EL protein via alterations in transcriptional efficiency. Specific Aim 1: To determine if coding variants of EL increase HDL by decreasing EL activity. To test for decreased activity, coding variants will be recreated in an EL expression plasmid via site-directed mutagenesis, expressed in 293 cells, and their specific activities assayed in vitro against synthetic phospholipids substrates and native HDL. To test for an influence on HDL levels in vivo, the coding variants will be cloned into AAV-based vectors, injected into EL-KO mice, and HDL levels assessed over 6 weeks. Association of the variants with human HDL levels will be tested statistically by comparing frequencies among subjects from HDL extremes and by analyzing cosegregation with HDL levels within families. Specific Aim 2: To determine if promoter variants of EL modulate HDL levels through their influence on the rate of transcription of the EL gene. To test for altered rates of transcription, promoter variants will be recreated via site-directed mutagenesis in a firefly luciferase expression plasmid driven by the EL promoter, and transfected into HUVEC cells. Expression results will be validated by allele-specific HaploChIP analysis on endothelial cells isolated from heterozygous subjects. Association of the variants with human HDL levels will be tested statistically by comparing frequencies among subjects from HDL extremes and by analyzing cosegregation with HDL levels within families. HDL levels correlate with protection from coronary heart disease, the leading cause of mortality among men and women. This proposal will attempt to determine the significance of endothelial lipase in human HDL metabolism, and validate it as a target for pharmacologic inhibition to raise HDL levels.